QC Report

	general
Report generated at	2025-02-22 03:03:27
Title	Liver
Description	ATAC-seq data from Liu et al. liver data processed in 2025
Pipeline version	v2.2.0
Pipeline type	atac
Genome	mm10
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2
Total Reads	137766628	138223394
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	134745158	135277039
Mapped Reads (QC-failed)	0	0
% Mapped Reads	97.8	97.89999999999999
Paired Reads	137766628	138223394
Paired Reads (QC-failed)	0	0
Read1	68883314	69111697
Read1 (QC-failed)	0	0
Read2	68883314	69111697
Read2 (QC-failed)	0	0
Properly Paired Reads	131897358	132182046
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	95.7	95.6
With itself	134451308	134992754
With itself (QC-failed)	0	0
Singletons	293850	284285
Singletons (QC-failed)	0	0
% Singleton	0.2	0.2
Diff. Chroms	123205	93298
Diff. Chroms (QC-failed)	0	0

Marking duplicates (filtered BAM)

	rep1	rep2
Unpaired Reads	0	0
Paired Reads	58680377	58291029
Unmapped Reads	0	0
Unpaired Duplicate Reads	0	0
Paired Duplicate Reads	3927859	3419112
Paired Optical Duplicate Reads	0	0
% Duplicate Reads	6.6936	5.8656

Filtered with samtools flag 1804 (samtools view -F 1804):

read unmapped (0x4)
mate unmapped (0x8, for paired-end)
not primary alignment (0x100)
read fails platform/vendor quality checks (0x200)
read is PCR or optical duplicate (0x400)

Fraction of mitochondrial reads (unfiltered BAM)

	rep1	rep2
Rn = Number of Non-mitochondrial Reads	134092785	134669768
Rm = Number of Mitochondrial Reads	807975	748982
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.0059894028766035124	0.005530858909862925

SAMstat (filtered/deduped BAM)

	rep1	rep2
Total Reads	109277812	109525878
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	109277812	109525878
Mapped Reads (QC-failed)	0	0
% Mapped Reads	100.0	100.0
Paired Reads	109277812	109525878
Paired Reads (QC-failed)	0	0
Read1	54638906	54762939
Read1 (QC-failed)	0	0
Read2	54638906	54762939
Read2 (QC-failed)	0	0
Properly Paired Reads	109277812	109525878
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	100.0	100.0
With itself	109277812	109525878
With itself (QC-failed)	0	0
Singletons	0	0
Singletons (QC-failed)	0	0
% Singleton	0.0	0.0
Diff. Chroms	0	0
Diff. Chroms (QC-failed)	0	0

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

	rep1	rep2
Fraction of reads in NFR	0.3390878900296575	0.41301047624114595
Fraction of reads in NFR (QC pass)	False	True
Fraction of reads in NFR (QC reason)	out of range [0.4, inf]	OK
NFR / mono-nuc reads	0.5574878496313785	0.7639725034037768
NFR / mono-nuc reads (QC pass)	False	False
NFR / mono-nuc reads (QC reason)	out of range [2.5, inf]	out of range [2.5, inf]
Presence of NFR peak	True	True
Presence of Mono-Nuc peak	True	True
Presence of Di-Nuc peak	True	True

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.

NFR: Nucleosome free region

Sequence quality metrics (filtered/deduped BAM)

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.

Annotated genomic region enrichment

	rep1	rep2
Fraction of Reads in universal DHS regions	0.4295542081314732	0.4208899562530784
Fraction of Reads in blacklist regions	0.003598223580830846	0.0034514948147687983
Fraction of Reads in promoter regions	0.07073438659258661	0.06644051737252452
Fraction of Reads in enhancer regions	0.35549647535036666	0.3512251323837824

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.

Library complexity quality metrics

Library complexity (filtered non-mito BAM)

	rep1	rep2
Total Fragments	58447808	58074103
Distinct Fragments	54700569	54824302
Positions with Two Read	2902365	2513223
NRF = Distinct/Total	0.935887	0.94404
PBC1 = OneRead/Distinct	0.942972	0.950882
PBC2 = OneRead/TwoRead	17.772091	20.742868

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.

Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally

0-0.5 is severe bottlenecking
0.5-0.8 is moderate bottlenecking
0.8-0.9 is mild bottlenecking
0.9-1.0 is no bottlenecking

Replication quality metrics

IDR (Irreproducible Discovery Rate) plots

Reproducibility QC and peak detection statistics

	overlap	idr
Nt	193004	133410
N1	150445	104006
N2	139858	94177
Np	193846	135050
N optimal	193846	135050
N conservative	193004	133410
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.0043626038838573	1.0122929315643505
Self Consistency Ratio	1.0756982081825852	1.1043673083661616
Reproducibility Test	pass	pass

Reproducibility QC

N1: Replicate 1 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep1 reads)
N2: Replicate 2 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep2 reads)
Ni: Replicate i self-consistent peaks (comparing two pseudoreplicates generated by subsampling RepX reads)
Nt: True Replicate consistent peaks (comparing true replicates Rep1 vs Rep2)
Np: Pooled-pseudoreplicate consistent peaks (comparing two pseudoreplicates generated by subsampling pooled reads from Rep1 and Rep2)
Self-consistency Ratio: max(N1,N2) / min (N1,N2)
Rescue Ratio: max(Np,Nt) / min (Np,Nt)
Reproducibility Test: If Self-consistency Ratio >2 AND Rescue Ratio > 2, then 'Fail' else 'Pass'

Number of raw peaks

	rep1	rep2
Number of peaks	264873	258482

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	150.0	150.0	150.0	150.0
25 percentile	224.0	210.0	454.0	375.0
50 percentile (median)	350.0	324.0	691.0	566.0
75 percentile	624.0	582.0	1016.0	875.0
Max size	3322.0	2554.0	3471.0	3471.0
Mean	479.5627678170293	450.2582384846914	775.3431988152536	669.8014403186035

Enrichment / Signal-to-noise ratio

TSS enrichment (filtered/deduped BAM)

	rep1	rep2
TSS enrichment	7.692382151686117	7.354598361825624

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/

Jensen-Shannon distance (filtered/deduped BAM)

	rep1	rep2
AUC	0.26416786377332707	0.27191882248032284
Synthetic AUC	0.49546711027448853	0.49546942218720447
X-intercept	0.09841671032477635	0.09805478126287248
Synthetic X-intercept	0.0	0.0
Elbow Point	0.6291026958716092	0.6162232202488312
Synthetic Elbow Point	0.50623095733528	0.506253689441241
Synthetic JS Distance	0.3184199050659078	0.3046303531126913

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep2	rep1-pr1	rep2-pr1	rep1-pr2	rep2-pr2	pooled	pooled-pr1	pooled-pr2
Fraction of Reads in Peaks	0.18865671468605172	0.1734038324714457	0.17115146851585938	0.15774441620555799	0.171099564109135	0.15723451506564531	0.19181610694042683	0.1815989741160309	0.1817044418373789

FRiP for overlap peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.16278012039010858	0.15235090907566853	0.13647040565153012	0.16303710417315173

FRiP for IDR peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.13961006781924015	0.12725294133817394	0.11198888540295472	0.14051592091522772

For macs2 raw peaks:

repX: Peak from true replicate X
repX-prY: Peak from Yth pseudoreplicates from replicate X
pooled: Peak from pooled true replicates (pool of rep1, rep2, ...)
pooled-pr1: Peak from 1st pooled pseudo replicate (pool of rep1-pr1, rep2-pr1, ...)
pooled-pr2: Peak from 2nd pooled pseudo replicate (pool of rep1-pr2, rep2-pr2, ...)

For overlap/IDR peaks:

repX_vs_repY: Comparing two peaks from true replicates X and Y
repX-pr1_vs_repX-pr2: Comparing two peaks from both pseudoreplicates from replicate X
pooled-pr1_vs_pooled-pr2: Comparing two peaks from 1st and 2nd pooled pseudo replicates